PURIFICATION OF CATALYTIC DOMAIN OF RAT SPLEEN P72(SYK) KINASE AND ITS PHOSPHORYLATION AND ACTIVATION BY PROTEIN-KINASE-C

The catalytic domain of p72(syk) kinase (CDp72(syk)) was purified from a 30 000 g particulate fraction of rat spleen. The purification proce dure employed sequential chromatography on columns of DEAE-Sephacel an d Superdex-200, and elution from HA-Ultrogel by chloride. The analysis of the final CDp72(syk) preparation by SDS/PAGE revealed a major silv er-stained 40 kDa protein. The kinase was identified by covalent modif ication of its ATP-binding site with [C-14]5'-fluorosulphonylbenzoylad enosine and by immunoblotting with a polyclonal antibody against the ' linker' region of p72(syk). By using poly(Glu(4), Tyr(1)) as a substra te, the specific activity of the enzyme was determined as 18.5 nmol P- i/min per mg. Casein, histones H1 and H2B and myelin basic protein wer e efficiently phosphorylated by CDp72(syk). The kinase exhibited a lim ited ability to phosphorylate random polymers containing tyrosine resi dues. CDp72(syk) autophosphorylation activity was associated with an a ctivation of the kinase towards exogenous substrates. The extent of ac tivation was dependent on the substrates added. CDp72(syk) was phospho rylated by protein kinase C (PKC) on serine and threonine residues. Wi th a newly developed assay method, we demonstrated that the PKC-mediat ed phosphorylation had a strong activating effect on the tyrosine kina se activity of CDp72(syk). Studies extended to conventional PKC isofor ms revealed an isoform-dependent manner (alpha > beta(1) = beta(II) > gamma) of CDp72(syk) phosphorylation. The different phosphorylation ef ficiencies of the PKC isoforms closely correlated with the ability to enhance the tyrosine kinase activity.